BirthSeq, a new method to isolate and analyze dated cells in different vertebrates.

Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well documented, with evidence showing that time plays a crucial role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via fluorescence-activated cell sorting, and analysis using high-throughput techniques. We calibrated the BirthSeq method for developmental organs across three vertebrate species (mouse, chick and gecko), and utilized it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organisms, aiding developmental biology research by targeting cells and their temporal cues.

Lymphocyte Subsets in Cervicovaginal Lavage Specimens of HIV-Infected Women: A Surrogate Risk Marker of HPV-Associated Cervical Lesions.

INTRODUCTION: To better understand the role of mucosa immunity in the development of cervical carcinoma in HIV infection, cervical lymphocyte subsets were characterized in HIV+ and HIV- women, as well as their relation to HPV-associated cervical lesions. METHODS: Eighty-three (52 HIV+, 31 HIV-) cell suspensions of cervicovaginal lavage (CVL) and 52 HIV+ peripheral blood (PB) samples were assessed by flow cytometry to evaluate lymphoid populations. High-risk (HR) HPV was assessed in liquid-based cytology and HIV mRNA in PB in the same patients. RESULTS: Cervical CD4+ T cells and CD4+/CD8+ ratio were decreased (p < 0.0001) and cervical CD8+ T cells were increased (p = 0.0080) in HIV+ women. These patients had lower CD4+ T-cell percentages in CVL compared to PB (p = 0.0257), and the opposite was true for CD8+ T cells (p = 0.0104). They also had a higher prevalence of high-grade squamous intraepithelial lesions (SILs) with an increased prevalence of HR HPV. Cervical CD8+ T cells were increased in HR HPV+ patients (p = 0.0300) and related to higher prevalence of SILs (p = 0.0001). DISCUSSION/CONCLUSION: Cervical lymphoid populations can be characterized by flow cytometry, showing a distinct cervical T-cell compartment in HIV+ women. This may represent a surrogate risk marker of HPV-associated cervical lesions in this population and prompt further research on this subject, contributing to improving patients' management.

Keratoconus tomographic indices in osteogenesis imperfecta.

PURPOSE: Osteogenesis imperfecta (OI) is a rare inherited disease affecting collagen-rich tissues. Ocular complications have been reported such as thin corneas, low ocular rigidity, keratoconus, among others. The purpose of this study is to characterize corneal tomographic features in OI patients compared to unaffected patients, with particular focus on commonly studied keratoconus indices. METHODS: Cross-sectional case-control study including 37 OI patients and 37 age-matched controls. Patients and controls underwent comprehensive ophthalmological examination including corneal Scheimpflug tomography with a Pentacam HR device (Oculus Optikgeräte GmbH, Wetzlar, Germany) to analyse and compare topometric, tomographic, pachymetric and Belin-Ambrósio Enhanced Ectasia Display III (BAD-D) data of both eyes of each patient. RESULTS: Most OI patients had type I disease (n = 24; 65%) but type III-VII patients were also included. Two patients had clinically overt bilateral keratoconus. OI patients had significantly higher maximum keratometry (45.2 ± 2.1 vs. 43.7 ± 1.2; p = 0.0416), front and back elevation (3.0 ± 3.3 vs. 2.1 ± 1.3, p = 0.0201; 11.1 ± 8.2 vs. 5.0 ± 3.7, p < 0.0001), index of surface variance (25.5 ± 13 vs. 17.4 ± 8.3; p = 0.0016), index of vertical asymmetry (0.21 ± 0.14 vs. 0.15 ± 0.06; p = 0.0215), index of height asymmetry (9.2 ± 14 vs. 6.0 ± 4.5; p = 0.0421), index of height decentration (0.02 ± 0.01 vs. 0.01 ± 0.01; p < 0.0001) and average pachymetric progression (1.01 ± 0.19 vs. 0.88 ± 0.14; p < 0.0001) readings. Thinnest corneal thickness and maximum Ambrósio relational thickness were significantly lower (477 ± 52 vs. 543 ± 26; 387 ± 95 vs. 509 ± 49; p < 0.0001). Two-thirds of OI patients had corneas with a minimum thickness < 500 µm. BAD-D value was significantly higher in OI patients (2.1 ± 1.4 vs. 0.9 ± 0.2; p < 0.0001). CONCLUSION: OI patients showed significant changes in corneal profiles compared with healthy subjects. A high proportion of patients had tomographically suspect corneas when using keratoconus diagnostic indices. Further studies are warranted to assess the true risk of corneal ectasia in OI patients.

Detection of HIV mRNA in routine liquid-based cytology specimens of HIV-infected women.

OBJECTIVE: Human immunodeficiency virus-infected women have a high incidence of HPV infection, and HIV and HPV coinfection is associated with high incidence of cervical intraepithelial lesions and cervical cancer. This study investigated the ability to detect HIV mRNA in routine screening cervical liquid-based cytology (LBC) samples and its correlation with HPV coinfection and cervical intraepithelial lesions. METHODS: Liquid-based cytology samples from 80 HIV-infected women under combined antiretroviral therapy (cART) were studied for detection of HIV and HPV mRNA using Aptima® tests and for cytology diagnosis according to the 2014 Bethesda System for Reporting Cervical Cytology. Peripheral blood (PB) HIV mRNAs were assessed by real-time polymerase chain reaction (RT-PCR). Statistical analysis used Fisher's exact or Chi-square test to compare frequencies among groups and the Mann-Whitney U test to compare continuous variables. RESULTS: Human immunodeficiency virus mRNA was present in 21.3% of routine LBC samples in HIV-infected women, 12.5% of which had no detectable PB viral load. Among 10 patients diagnosed with high-grade squamous intraepithelial lesion (HSIL), 50% had detectable HIV viral load. The occurrence of HSIL vs low-grade intraepithelial lesion/negative intraepithelial lesion or malignancy in LBC samples was significantly higher in women with detectable HIV viral load (P = .029). CONCLUSIONS: Human immunodeficiency virus mRNA was present in routine LBC samples in HIV-positive women under cART. Detection of HIV viral load in LBC is significantly associated with cervical HSIL. This suggests the relevance of HIV mRNA viral load assessment in routine LBC, to evaluate patients' infectious potential and monitor efficacy of the cART scheme.

Characterization of the African Swine Fever Virus Decapping Enzyme during Infection.

African swine fever virus (ASFV) infection is characterized by a progressive decrease in cellular protein synthesis with a concomitant increase in viral protein synthesis, though the mechanism by which the virus achieves this is still unknown. Decrease of cellular mRNA is observed during ASFV infection, suggesting that inhibition of cellular proteins is due to an active mRNA degradation process. ASFV carries a gene (Ba71V D250R/Malawi g5R) that encodes a decapping protein (ASFV-DP) that has a Nudix hydrolase motif and decapping activity in vitro Here, we show that ASFV-DP was expressed from early times and accumulated throughout the infection with a subcellular localization typical of the endoplasmic reticulum, colocalizing with the cap structure and interacting with the ribosomal protein L23a. ASFV-DP was capable of interaction with poly(A) RNA in cultured cells, primarily mediated by the N-terminal region of the protein. ASFV-DP also interacted with viral and cellular RNAs in the context of infection, and its overexpression in infected cells resulted in decreased levels of both types of transcripts. This study points to ASFV-DP as a viral decapping enzyme involved in both the degradation of cellular mRNA and the regulation of viral transcripts.IMPORTANCE Virulent ASFV strains cause a highly infectious and lethal disease in domestic pigs for which there is no vaccine. Since 2007, an outbreak in the Caucasus region has spread to Russia, jeopardizing the European pig population and making it essential to deepen knowledge about the virus. Here, we demonstrate that ASFV-DP is a novel RNA-binding protein implicated in the regulation of mRNA metabolism during infection, making it a good target for vaccine development.

African swine fever virus uses macropinocytosis to enter host cells.

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

Programmed death-ligand 1 (PD-L1) expression in cervical intraepithelial neoplasia and cervical squamous cell carcinoma of HIV-infected and non-infected patients.

Programmed death-ligand 1 (PD-L1) is overexpressed in cervical carcinoma, hindering tumor destruction. The aim of this study was to assess PD-L1 expression by immunohistochemistry in cervical squamous cell carcinoma (SCC) and squamous intraepithelial lesions (SILs) from human immunodeficiency virus-positive (HIV+) and human immunodeficiency virus-negative (HIV-) patients. A total of 166 SCC and SIL samples of HIV+ and HIV- patients were included and analyzed for PD-L1 expression through tumor proportion score (TPS), and results were stratified in five TPS groups using SP263 antibody and, combined positive score (CPS) using 22C3 antibody. In cohort 1 (SP263 clone), all HIV+ patients were negative for intraepithelial lesion or malignancy (NILM), and low-grade squamous intraepithelial lesions (LSILs) scored < 1; and 87.5% of high-grade squamous intraepithelial lesions (HSILs) adjacent to SCC, 19% of HSILs non-adjacent to SCC, and 69% of SCCs scored ≥ 1 (15.4% scored 5). In HIV- patients, all NILM, LSILs, HSILs adjacent to SCC, and two HSILs non-adjacent to SCC scored < 1. SCC: 88.2% scored ≥ 1 and 5.9% scored 5. In cohort 2 (SP263 and 22C3 clones), 16.7% of HIV+ patients with SCC were positive with both clones, CPS ≥ 1 (22C3) or score 5 (≥ 50%) (SP263), showing no significant differences in positivity between both clones. These results indicate that a relatively low percentage of SCCs (16.7%; both in HIV+ and in HIV- patients) express PD-L1 (TPS ≥ 50% and CPS > 1), which may be due to some samples being archival material, sample characteristics, or use of different methodologies, highlighting the need for standardization of PD-L1 assessment in SCC of the cervix. The fact that PD-L1 is overexpressed in SILs of HIV+ patients suggests potential additional applications for immunotherapy in this disease.

C1-C2 Transarticular Anterior Fixation for the Treatment of Atlantoaxial Traumatic Dislocation: A Clinical Case.

Fractures of the odontoid apophysis are one of the most frequent lesions in the elderly population, and an increasingly preponderant problem with the progressive aging of the world population. In the present work, we report a clinical case of an 88-year-old male patient who suffered a fall resulting in a type-II fracture of the odontoid apophysis on the Anderson-D'Alonzo classification. Given the age and comorbidities of the patient, we decided to perform osteosynthesis of the fracture through anterior fixation with a transarticular screw in combination with fixation with an odontoid screw. This technique enables the necessary stability for the consolidation of Anderson-D'Alonzo's type II odontoid apophysis fracture, with the advantage of the lower levels of dissection of the cervical extensor musculature and hemorrhage resulting from this aggression when compared with the posterior approach; moreover, it is a readily-available technique that yields clear benefits in the treatment of this pathology in the geriatric population.

Asymptomatic chronic red eye: A surgical technique case report.

Introduction and importance: Ocular Surface Squamous-cell Neoplasia (OSSN) is an infrequent diagnosis whose clinical suspicion assumes great importance and should not be overlooked. The following case-report aims to describe the diagnosis and treatment of a patient with OSSN whose complaints were mild in comparison to the severity of the disease. The chosen surgical technique was paramount for a disease-free outcome while minimizing the scarring effects of surgical removal. CASE PRESENTATION: Patient presented mild discomfort right eye and painless persistent hyperaemia. Slit-lamp observation showed a clear diagnosis and lesion's extent evaluated through multimodal imaging. After surgical excision the patient underwent topical ocular treatment with mitomycin-C for a higher margin of safety even before the pathology results were available. DISCUSSION: Ancillary exam technology improvement has allowed a higher margin of safety while determining the extent of OSSN lesions. In the absence of clear diagnostic criteria and guidelines, clinical reasoning and OSSN awareness are critical for timely diagnosis and treatment, as several treatment options are available, allowing an increasing number of patients to be treated non-invasively. In this case-report, we highlight the importance of early-recognition and the reasoning for choosing a combined treatment option with a higher margin of safety. CONCLUSION: Early recognition and prompt treatment of OSSN lesions is of paramount importance to avoid ocular invasiveness and potentially preclude both ocular and systemic complication. The choice of a combined surgical and medical approach may provide a higher margin of safety for suitable cases. This patient is currently disease-free at 6-month follow-up.

Clonal hematopoiesis is not prevalent in Hutchinson-Gilford progeria syndrome.

Clonal hematopoiesis of indeterminate potential (CHIP), defined as the presence of somatic mutations in cancer-related genes in blood cells in the absence of hematological cancer, has recently emerged as an important risk factor for several age-related conditions, especially cardiovascular disease. CHIP is strongly associated with normal aging, but its role in premature aging syndromes is unknown. Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare genetic condition driven by the accumulation of a truncated form of the lamin A protein called progerin. HGPS patients exhibit several features of accelerated aging and typically die from cardiovascular complications in their early teens. Previous studies have shown normal hematological parameters in HGPS patients, except for elevated platelets, and low levels of lamin A expression in hematopoietic cells relative to other cell types in solid tissues, but the prevalence of CHIP in HGPS remains unexplored. To investigate the potential role of CHIP in HGPS, we performed high-sensitivity targeted sequencing of CHIP-related genes in blood DNA samples from a cohort of 47 HGPS patients. As a control, the same sequencing strategy was applied to blood DNA samples from middle-aged and elderly individuals, expected to exhibit a biological age and cardiovascular risk profile similar to HGPS patients. We found that CHIP is not prevalent in HGPS patients, in marked contrast to our observations in individuals who age normally. Thus, our study unveils a major difference between HGPS and normal aging and provides conclusive evidence that CHIP is not frequent in HGPS and, therefore, is unlikely to contribute to the pathophysiology of this accelerated aging syndrome.

Pelvic Desmoid Tumor: A Rare Case with Difficult Diagnosis and Treatment.

Desmoid tumors are rare benign neoplasms, with locally aggressive characteristics. Ongoing or previous pregnancy, antecedent trauma, and familial adenomatous polyposis are known risk factors. Still, the majority of cases are sporadic and its etiology is still unknown. These tumors may occur in any body site, but retroperitoneal and pelvic desmoid tumors are extremely rare. Nonspecific clinical and radiological findings lead to erroneous diagnosis in 50% of patients before surgery. We present a case of a young multiparous female with a deep infiltrative lesion adherent to the right pelvic sidewall leading to severe right hydroureteronephrosis and ipsilateral loss of renal function. Although deep endometriosis was suspected, malignancy features could not be excluded by imaging studies. The patient underwent an exploratory laparotomy for definite diagnosis and treatment, which led to right nephrectomy, hysterectomy, and right oophorectomy because of deep infiltration and difficult dissection. Definite histologic diagnosis revealed the presence of a pelvic desmoid tumor. Positive margins were encountered but, until this moment, no disease relapse occurred.

Topical corticosteroids with topical cyclosporine A versus topical corticosteroids alone for immunological corneal graft rejection.

PURPOSE: To assess the efficacy and safety of supplementing topical cyclosporine A (CsA) to topical corticosteroids (CS), in the prophylaxis and treatment of corneal graft rejection following penetrating keratoplasty (PK). METHODS: Meta-analysis. Search was performed in PubMed, CENTRAL, ClinicalTrials.gov, reference lists of articles and conference proceedings. Primary outcomes: 1-year rejection-free survival rate (prophylaxis); resolution rate of rejection episodes (treatment). Secondary outcomes: 6- and 24-month rejection-free graft survival rate, number of rejection episodes during follow-up, time-to-resolution of rejection episode, 12- and 24-months graft survival rate, adverse events. Subgroup analyses were planned for high-risk grafts; primary vs. secondary prophylaxis of graft rejection episodes; and CsA concentrations of 0.05%, 1%, and 2%. RESULTS: Five studies of moderate methodological quality were included (one retrospective, four RCT), assessing 459 eyes (CS + CsA 226, CS 233). In the prophylaxis setting, supplemental CsA was associated with a higher rejection-free survival rate at 12-months (RR 1.25, 95% CI: 1.00-1.56, p = 0.05) and 24-months post-PK (RR 1.56, 95% CI: 1.15-2.11, p < 0.01), though no differences were found at the 6-months timepoint (p = 0.93). This effect was mostly verified using CsA 2% in the high-risk subset of patients. In the treatment setting, no differences were found in the resolution rate of rejection episodes (p = 0.23). No differences existed on drug-related adverse events. CONCLUSION: In the prophylaxis of rejection episodes post-PK, the combined regimen of CS + CsA was associated with a higher 1- and 2-year rejection-free graft survival rate. Subgroup analysis mostly supported the use of CsA 2% for high-risk grafts. Further studies are needed to validate these results.

Trained immunity induction by the inactivated mucosal vaccine MV130 protects against experimental viral respiratory infections.

MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity.

In vivo expansion of a CD9+ decidual-like NK cell subset following autologous hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a treatment option for hematological disorders and pediatric solid tumors. After an autoHSCT, natural killer (NK) cells are the first lymphocyte subset returning to normal levels. To uncover global changes during NK cell reconstitution after autoHSCT, we performed RNA-sequencing on NK cells before and after autoHSCT. Results showed profound changes in the gene expression profile of NK cells immediately after autoHSCT. Several biological processes including cell cycle, DNA replication and the mevalonate pathway were enriched. Significantly, we observed that following autoHSCT, NK cells acquired a decidual-like gene expression profile, including the expression of CD9. By using multiparametric flow cytometry, we confirmed the expansion of NK cells expressing CD9 immediately after autoHSCT, which exhibited higher granzyme B and perforin expression levels than CD9- NK cells. These results provide insights into the physiopathology of NK cells during their reconstitution after autoHSCT.

Regulation of host translational machinery by African swine fever virus.

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2alpha, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.

Relative Age Effect in Physical Fitness of South Portugal Students between 10 and 18 Years Old.

Relative age is a phenomenon broadly studied in sport sciences. Youth sports participants born earlier in the selection year tend to present a maturational advantage over their peers. As it is also dependent on physical performance, older physical education students may also benefit from this effect in this school subject. The main goal of this manuscript was to determine whether the relative age effect is present within physical fitness outcomes of Portuguese children and adolescents. The physical-aerobic fitness, strength, flexibility and body composition of 885 students (490 females and 395 males) were collected and compared by quarters of birth, segmented by gender and age groups (10-12; 12-14; 14-16 and 16-18 years). The results reveal a moderate to small effect in physical fitness outcomes, with a trend for children and adolescents born in the early part of the year to present higher performance levels. These differences were more evident in ages closer to the physical maturational onset (12-14 y) and more apparent in male students. This physical fitness advantage may lead to a biased assessment and development of students born earlier in the year.

CD4+ and CD8+ cell populations in HIV-positive women with cervical squamous intra-epithelial lesions and squamous cell carcinoma.

INTRODUCTION: This study aimed to analyse cervical lymphocytic populations in HIV+ and HIV- patients and correlate different cervical lesions with HIV viral load and presence of high-risk HPV types. MATERIAL AND METHODS: A total of 132 histological specimens from 40 HIV+ and 72 HIV- patients were evaluated for CD4+ and CD8+ T cell distribution, presence of high-risk HPV types, peripheral blood HIV viral load and CD4+/CD8+ ratio. RESULTS: High-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) from HIV+ patients had lower CD4+ T cell scores compared with HIV- patients. In all lesion groups, HIV+ patients presented higher epithelial and stromal CD8+ T cell scores. HIV viral load was more often detectable in patients with SCC than in those with low-grade squamous intraepithelial lesion (LSIL) (p = 0.0409). HSIL HIV+ patients had lower circulating CD4+ T cell counts (p = 0.0434) and CD4+/CD8+ ratio (p = 0.0378) compared with LSIL HIV+ patients. High-risk HPV types other than 16 and 18/45 were more prevalent in the HIV+ group. DISCUSSION: These results support an imbalance between cervical CD4+ and CD8+ T lymphocytes of HIV+ patients with SIL and SCC, with increased CD8+ infiltrate density with lesion severity, even in patients with immune system recovery under cART.

Clonal Hematopoiesis and Risk of Progression of Heart Failure With Reduced Left Ventricular Ejection Fraction.

BACKGROUND: Clonal hematopoiesis driven by somatic mutations in hematopoietic cells, frequently called clonal hematopoiesis of indeterminate potential (CHIP), has been associated with adverse cardiovascular outcomes in population-based studies and in patients with ischemic heart failure (HF) and reduced left ventricular ejection fraction (LVEF). Yet, the impact of CHIP on HF progression, including nonischemic etiology, is unknown. OBJECTIVES: The purpose of this study was to assess the clinical impact of clonal hematopoiesis on HF progression irrespective of its etiology. METHODS: The study cohort comprised 62 patients with HF and LVEF <45% (age 74 ± 7 years, 74% men, 52% nonischemic, and LVEF 30 ± 8%). Deep sequencing was used to detect CHIP mutations with a variant allelic fraction >2% in 54 genes. Patients were followed for at least 3.5 years for various adverse events including death, HF-related death, and HF hospitalization. RESULTS: CHIP mutations were detected in 24 (38.7%) patients, without significant differences in all-cause mortality (p = 0.151). After adjusting for risk factors, patients with mutations in either DNA methyltransferase 3 alpha (DNMT3A) or Tet methylcytosine dioxygenase 2 (TET2) exhibited accelerated HF progression in terms of death (hazard ratio [HR]: 2.79; 95% confidence interval [CI]: 1.31 to 5.92; p = 0.008), death or HF hospitalization (HR: 3.84; 95% CI: 1.84 to 8.04; p < 0.001) and HF-related death or HF hospitalization (HR: 4.41; 95% CI: 2.15 to 9.03; p < 0.001). In single gene-specific analyses, somatic mutations in DNMT3A or TET2 retained prognostic significance with regard to HF-related death or HF hospitalization (HR: 4.50; 95% CI: 2.07 to 9.74; p < 0.001, for DNMT3A mutations; HR: 3.18; 95% CI: 1.52 to 6.66; p = 0.002, for TET2 mutations). This association remained significant irrespective of ischemic/nonischemic etiology. CONCLUSIONS: Somatic mutations that drive clonal hematopoiesis are common among HF patients with reduced LVEF and are associated with accelerated HF progression regardless of etiology.

Alterations in Circulating Monocytes Predict COVID-19 Severity and Include Chromatin Modifications Still Detectable Six Months after Recovery.

An early analysis of circulating monocytes may be critical for predicting COVID-19 course and its sequelae. In 131 untreated, acute COVID-19 patients at emergency room arrival, monocytes showed decreased surface molecule expression, including low HLA-DR, in association with an inflammatory cytokine status and limited anti-SARS-CoV-2-specific T cell response. Most of these alterations had normalized in post-COVID-19 patients 6 months after discharge. Acute COVID-19 monocytes transcriptome showed upregulation of anti-inflammatory tissue repair genes such as BCL6, AREG and IL-10 and increased accessibility of chromatin. Some of these transcriptomic and epigenetic features still remained in post-COVID-19 monocytes. Importantly, a poorer expression of surface molecules and low IRF1 gene transcription in circulating monocytes at admission defined a COVID-19 patient group with impaired SARS-CoV-2-specific T cell response and increased risk of requiring intensive care or dying. An early analysis of monocytes may be useful for COVID-19 patient stratification and for designing innate immunity-focused therapies.

Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture.

The high complexity of naturally occurring microbial communities is the major drawback limiting the study of these important biological systems. In this study, a comparison between pure cultures of Pseudomonas reinekei sp. strain MT1 and stable community cultures composed of MT1 plus the addition of Achromobacter xylosoxidans strain MT3 (in a steady-state proportion 9:1) was used as a model system to study bacterial interactions that take place under simultaneous chemical and oxidative stress. Both are members of a real community isolated from a polluted sediment by enrichment in 4-chlorosalicylate (4CS). The analysis of dynamic states was carried out at the proteome, metabolic profile and population dynamic level. Differential protein expression was evaluated under exposure to 4CS and high concentrations of toxic intermediates (4-chlorocatechol and protoanemonin), including proteins from several functional groups and particularly enzymes of aromatic degradation pathways and outer membrane proteins. Remarkably, 4CS addition generated a strong oxidative stress response in pure strain MT1 culture led by alkyl hydroperoxide reductase, while the community showed an enhanced central metabolism response, where A. xylosoxidans MT3 helped to prevent toxic intermediate accumulation. A significant change in the outer membrane composition of P. reinekei MT1 was observed during the chemical stress caused by 4CS and in the presence of A. xylosoxidans MT3, highlighting the expression of the major outer membrane protein OprF, tightly correlated to 4CC concentration profile and its potential detoxification role.